Coronaviruses(CoVs)are important human and animal pathogens that cause respiratory and gastrointestinal diseases.Porcine epidemic diarrhoea(PED),characterized by severe diarrhoea and vomiting in pigs,is a highly letha...Coronaviruses(CoVs)are important human and animal pathogens that cause respiratory and gastrointestinal diseases.Porcine epidemic diarrhoea(PED),characterized by severe diarrhoea and vomiting in pigs,is a highly lethal disease caused by porcine epidemic diarrhoea virus(PEDV)and causes substantial losses in the swine industry worldwide.However,currently available commercial drugs have not shown great therapeutic effects.In this study,a fluorescence resonance energy transfer(FRET)-based assay was applied to screen a library containing 1,590 compounds and identified two compounds,3-(aminocarbonyl)-1-phenylpyridinium and 2,3-dichloronaphthoquinone,that target the 3C-like protease(3CL^(pro))of PEDV.These compounds are of low molecular weight(MW)and greatly inhibited the activity of this enzyme(IC_(50) values were obtained in this study).Furthermore,these compounds exhibited antiviral capacity against another member of the CoV family,feline infectious peritonitis virus(FIPV).Here,the inhibitory effects of these compounds against CoVs on Vero cells and feline kidney cells were identified(with EC_(50) values)and cell viability assays were performed.The results of putative molecular docking models indicate that these compounds,labeled compound 1 and compound 2,contact the conserved active sites(Cys144,Glu165,Gln191)of 3CL^(pro) via hydrogen bonds.These findings provide insight into the antiviral activities of compounds 1 and 2 that may facilitate future research on anti-CoV drugs.展开更多
The coronavirus 3C-like(3CL)protease,a cysteine protease,plays an important role in viral infection and immune escape.However,there is still a lack of effective tools for determining the cleavage sites of the 3CL prot...The coronavirus 3C-like(3CL)protease,a cysteine protease,plays an important role in viral infection and immune escape.However,there is still a lack of effective tools for determining the cleavage sites of the 3CL protease.This study systematically investigated the diversity of the cleavage sites of the coronavirus 3CL protease on the viral polyprotein,and found that the cleavage motif were highly conserved for viruses in the genera of Alphacoronavirus,Betacoronavirus and Gammacoronavirus.Strong residue preferences were observed at the neighboring positions of the cleavage sites.A random forest(RF)model was built to predict the cleavage sites of the coronavirus 3CL protease based on the representation of residues in cleavage motifs by amino acid indexes,and the model achieved an AUC of 0.96 in cross-validations.The RF model was further tested on an independent test dataset which were composed of cleavage sites on 99 proteins from multiple coronavirus hosts.It achieved an AUC of 0.95 and predicted correctly 80%of the cleavage sites.Then,1,352 human proteins were predicted to be cleaved by the 3CL protease by the RF model.These proteins were enriched in several GO terms related to the cytoskeleton,such as the microtubule,actin and tubulin.Finally,a webserver named 3CLP was built to predict the cleavage sites of the coronavirus 3CL protease based on the RF model.Overall,the study provides an effective tool for identifying cleavage sites of the 3CL protease and provides insights into the molecular mechanism underlying the pathogenicity of coronaviruses.展开更多
METTL3 is a crucial mRNA methyltransferase in mammals,essential for the regulation of gene expression and various biological processes.Here,we demonstrate that Mettl3 knockout(KO)in mouse embryonic stem cells(mESCs)le...METTL3 is a crucial mRNA methyltransferase in mammals,essential for the regulation of gene expression and various biological processes.Here,we demonstrate that Mettl3 knockout(KO)in mouse embryonic stem cells(mESCs)leads to widespread upregulation of transposable elements(TEs)and 2-cell(2C)-like genes in a m^(6)A enzyme activitydependent manner,independent of culture conditions.Furthermore,embryo chimera experiments using a transient METTL3 degradation system(dTAG)revealed that METTL3-deficient mESCs can contribute to trophectoderm lineages at the blastocyst stage,indicative of expanded developmental potential.These findings highlight the role of METTL3-mediated m^(6)A modifications in regulating the transcriptional and developmental plasticity of mESCs and suggest a link between m^(6)A loss and the acquisition of a 2C-like state with features of extended potency.展开更多
Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the causative agent of novel coronavirus disease 2019,can cause acute respiratory symptoms and even death globally.However,the immune escape mechanism and vi...Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the causative agent of novel coronavirus disease 2019,can cause acute respiratory symptoms and even death globally.However,the immune escape mechanism and viral pathogenesis remain poorly understood.Here,we report that the SARS-CoV-23C-like(3CL)protease specifically cleaves gasdermin D(GSDMD)at Q29 and Q193,producing two N-terminal fragments,GSDMD1-29 and GSDMD1-193.We also found that SARS-CoV-2 infection induced the cleavage of GSDMD.Then,we demonstrated that the ability to cleave GSDMD was dependent on the protease activity of the 3CL protease.Interestingly,unlike the GSDMD1-275 fragment cleaved by caspase-1,GSDMD1-29 and GSDMD1-193 did not trigger pyroptosis or inhibit SARS-CoV-2 replication.Additionally,various RNA viral proteases display different preferences for cleaving GSDMD at Q29 and Q193.Our findings reveal a mechanism by which SARS-CoV-2 and other RNA viruses inhibit pyroptosis,highlighting the critical role of the 3CL protease in immune evasion and viral replication.展开更多
基金This work was supported by the National Key R&D Plan of China(grant no.2018YFD0500102)the Natural Science Foundation of Hubei Province of China(grant no.2016CFA069)。
文摘Coronaviruses(CoVs)are important human and animal pathogens that cause respiratory and gastrointestinal diseases.Porcine epidemic diarrhoea(PED),characterized by severe diarrhoea and vomiting in pigs,is a highly lethal disease caused by porcine epidemic diarrhoea virus(PEDV)and causes substantial losses in the swine industry worldwide.However,currently available commercial drugs have not shown great therapeutic effects.In this study,a fluorescence resonance energy transfer(FRET)-based assay was applied to screen a library containing 1,590 compounds and identified two compounds,3-(aminocarbonyl)-1-phenylpyridinium and 2,3-dichloronaphthoquinone,that target the 3C-like protease(3CL^(pro))of PEDV.These compounds are of low molecular weight(MW)and greatly inhibited the activity of this enzyme(IC_(50) values were obtained in this study).Furthermore,these compounds exhibited antiviral capacity against another member of the CoV family,feline infectious peritonitis virus(FIPV).Here,the inhibitory effects of these compounds against CoVs on Vero cells and feline kidney cells were identified(with EC_(50) values)and cell viability assays were performed.The results of putative molecular docking models indicate that these compounds,labeled compound 1 and compound 2,contact the conserved active sites(Cys144,Glu165,Gln191)of 3CL^(pro) via hydrogen bonds.These findings provide insight into the antiviral activities of compounds 1 and 2 that may facilitate future research on anti-CoV drugs.
基金supported by the National Key Plan for Scientific Research and Development of China(2016YFD0500300)National Natural Science Foundation of China(32170651)Hunan Provincial Natural Science Foundation of China(2020JJ3006)。
文摘The coronavirus 3C-like(3CL)protease,a cysteine protease,plays an important role in viral infection and immune escape.However,there is still a lack of effective tools for determining the cleavage sites of the 3CL protease.This study systematically investigated the diversity of the cleavage sites of the coronavirus 3CL protease on the viral polyprotein,and found that the cleavage motif were highly conserved for viruses in the genera of Alphacoronavirus,Betacoronavirus and Gammacoronavirus.Strong residue preferences were observed at the neighboring positions of the cleavage sites.A random forest(RF)model was built to predict the cleavage sites of the coronavirus 3CL protease based on the representation of residues in cleavage motifs by amino acid indexes,and the model achieved an AUC of 0.96 in cross-validations.The RF model was further tested on an independent test dataset which were composed of cleavage sites on 99 proteins from multiple coronavirus hosts.It achieved an AUC of 0.95 and predicted correctly 80%of the cleavage sites.Then,1,352 human proteins were predicted to be cleaved by the 3CL protease by the RF model.These proteins were enriched in several GO terms related to the cytoskeleton,such as the microtubule,actin and tubulin.Finally,a webserver named 3CLP was built to predict the cleavage sites of the coronavirus 3CL protease based on the RF model.Overall,the study provides an effective tool for identifying cleavage sites of the 3CL protease and provides insights into the molecular mechanism underlying the pathogenicity of coronaviruses.
基金supported by the National Key Research and Development Program of China(2024YFA1107000,2024YFA1107901,2021YFA1102200 and 2024YFA1802300)the National Natural Science Foundation of China(32225012)+2 种基金Guangdong Basic and Applied Basic Research Foundation(2025A1515012713)partially supported by Science and Technology Planning Project of Guangdong Province,China(2023B1212060050,2023B1212120009)Health@InnoHK Program launched by Innovation Technology Commission of the Hong Kong SAR,China.
文摘METTL3 is a crucial mRNA methyltransferase in mammals,essential for the regulation of gene expression and various biological processes.Here,we demonstrate that Mettl3 knockout(KO)in mouse embryonic stem cells(mESCs)leads to widespread upregulation of transposable elements(TEs)and 2-cell(2C)-like genes in a m^(6)A enzyme activitydependent manner,independent of culture conditions.Furthermore,embryo chimera experiments using a transient METTL3 degradation system(dTAG)revealed that METTL3-deficient mESCs can contribute to trophectoderm lineages at the blastocyst stage,indicative of expanded developmental potential.These findings highlight the role of METTL3-mediated m^(6)A modifications in regulating the transcriptional and developmental plasticity of mESCs and suggest a link between m^(6)A loss and the acquisition of a 2C-like state with features of extended potency.
基金supported by the National Natural Science Foundation of China(82370015).
文摘Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),the causative agent of novel coronavirus disease 2019,can cause acute respiratory symptoms and even death globally.However,the immune escape mechanism and viral pathogenesis remain poorly understood.Here,we report that the SARS-CoV-23C-like(3CL)protease specifically cleaves gasdermin D(GSDMD)at Q29 and Q193,producing two N-terminal fragments,GSDMD1-29 and GSDMD1-193.We also found that SARS-CoV-2 infection induced the cleavage of GSDMD.Then,we demonstrated that the ability to cleave GSDMD was dependent on the protease activity of the 3CL protease.Interestingly,unlike the GSDMD1-275 fragment cleaved by caspase-1,GSDMD1-29 and GSDMD1-193 did not trigger pyroptosis or inhibit SARS-CoV-2 replication.Additionally,various RNA viral proteases display different preferences for cleaving GSDMD at Q29 and Q193.Our findings reveal a mechanism by which SARS-CoV-2 and other RNA viruses inhibit pyroptosis,highlighting the critical role of the 3CL protease in immune evasion and viral replication.
